Biolistic Transformation of Tetrahymena
This protocol applies to both vegetative and mated transformations. Day 1 Materials *DNAdel Gold carrier particles *Binding Buffer *Precipitation Buffer *EtOH (100%, chilled) *Sonicator *Plasmid DNA (linearized) *Macrocarriers (sterilized) *Macrocarrier holders (sterilized) *Starvation Buffer (10 mM Tris-HCl, pH 7.5) *Whatman-50 filter papers (sterilized) *Rupture Discs (900 psi or 300 psi rating) *Rupture Disc holder Method ''Coating Gold Particles with DNA #Sonicate the gold particles briefly to dissociate any aggregates prior to formulation. #The ratio of plasmid DNA to gold particles should be 2-5 µg of plasmid/mg of gold particles. A typical transformation experiment will utilise a mid-value of 3.5 µg of plasmid DNA/mg of gold particles. ''If 4 shots are planned (2.5 ''µ''g of plasmid DNA per shot), you will need a total of 10 µg of plasmid DNA. This means you'll need (10/3.85)=2.86 mg of gold particles. Seeing that the concentration of gold particles is 50 mg/ml, you'll pipette out (2.86/50)*1000=57 µL of gold particles. #Dilute the gold particles into binding buffer. To do this, use 20 µL of Binding Buffer for every 1.5 mg of gold particles. In our example above, we would dilute the gold particles using (2.86/1.5)*20=38 µL of Binding Buffer. #Immediately add linearized DNA. It is recommended to have the plasmid DNA at a concentration of 1 µg/µL. So 4 shots would require 10 ''µL of plasmid DNA (2.5 µg plasmid DNA per shot).'' #Add one volume of precipitation buffer to the total volume. Vortex briefly and let stand for 3 minutes on ice or at 4 °C. #Centrifuge at 10,000 rpm for 4-6 sec to pellet particles. Pipette out the supernatant carefully and add 1 mL of 100% cold EtOH. #Repeat centrifugation step and pipette out the supernatant. Then add 1 mL of 100% cold EtOH. #Repeat centrifugation step again and discard the supernatant. Harvest gold by resuspending in 100% ethanol. #Pipette up and down to resuspend and sonicate gold particles for 2-3 seconds in a bath sonicator to disrupt clumping. Working quickly, pipette 10 µL onto the center of each macrocarrier, assembled onto a macrocarrier holder, being sure to pipette up and down between removing each aliquot, as gold tends to settle quickly. #Place macrocarriers in Petridish containing drierite and place in a vacuum desiccant. Transport dried gold on macrocarrier from desiccator to the biolistic apparatus in a petridish containing drierite. #Lastly, place a circular Whatmann-50 filter in each of four 100-mm petridishes and moisten with Tris-HCl (~1 mL should work fine). This step is done just prior to the biolistic transformation. ''Checking Mating Efficiency If you are working with mated cells, check the mating efficiency to confirm that mating is successful and cultures are synchronous. #Check mated cells under the microscope at 2-3 hours for mating efficiency. If mating efficiency is less than 80%, then abort shooting experiment. Biolistic Transformation of Tetrahymena #Contamination can greatly reduce the transformation efficiency. Wipe the stage as well as hood area with 70% EtOH thoroughly. #Open the valve of the helium tank (turn in anticlockwise direction) and turn on the vacuum pump. #Wet one 900 psi rupture disc (or use three 300 psi rupture discs) in isopropanol, place in the rupture disc holder, and assemble onto the threaded helium port inside the particle bombardment apparatus. Make sure it is very tightly screwed on the port. #Harvest 50 mL of starved (or mating) cells by centrifugation at 1,100×''g for 1-1.5 min in a 50 mL conical tube. Decant the supernatant and distribute evenly onto filter paper, starting in the center and spreading cells radially outwards. #While cells are spinning, place the stopping screen on the bottom of the macrocarrier assembly. Then assemble the macrocarrier holder (with DNA-coated particles) and secure with lock ring and insert tray into the apparatus at highest position. #Place a petridish holding cells, with its lid removed, onto the specimen dish tray placed at the lowest position. #Turn on the vacuum compressor. Push the VACUUM switch and pull a vacuum to 26.5–27 mM Hg; once this value is reached on the pressure gauge, quickly switch VACUUM to HOLD. #Push and hold the ON button on the PRESSURE switch, record the pressure at which the rupture disc bursts delivering the coated particles in to cells below. #Release the ON button and switch VACUUM from HOLD to VENT. Once the vacuum is released, recover the petridish and add 1 mL of 10 mM Tris-HCl to the cells to prevent cells from drying out. #Place transformed cells in the 30 °C incubator and complete the remaining shots. #Allow the cells to recuperate for 10 minutes before transferring them to medium with appropriate drugs (2×SPP is used for mating cells). #Turn off vacuum pump and close helium tank valve (turn clockwise) when finished with biolistic transformations. Day 2 Materials *Selective Antibiotics *Multichannel Pipette *96-well plates *Petridishes Method ''Selection and Recovery of Transformants #If transforming vegetative cells for macronuclear transformants, then cells can be immediately returned to SPP growth medium. If the selectable marker used is expressed from cadmium inducible promoter, 0.5 mg/mL CdCl2 should be added to the medium during recovery to induce marker expression. #Cells can be selected ''en masse in flasks, but distributing at least part of the culture into 96-well plates allows recovery of individual transformants and assessment of transformation efficiency. #Place plates at 30 °C. Check for transformants at 3-5 days (depends on selective antibiotic use i.e. it takes closer to the 5 day mark to find resistance for Paromomycin with the K.O. construct) post-selection. Category:Transformation Category:Biolistic Category:Tetrahymena